Micro RNA (miRNA) is a small RNA comprising 20 to 23 bases and being untranslated into protein and, up to now, 1,000 or more kinds of miRNA have been identified on human genome. miRNA is produced in such a manner that, after transcribed from genome as primary miRNA (pri-miRNA) in several hundreds to several thousands of base length, it is cleaved by enzymes called Drosha and DGCR8 into a precursor miRNA (pre-miRNA) having stem loop structure comprising about 70 base pairs followed by treating with Dicer and TRBP/PACT whereupon miRNA is produced as a double-stranded RNA comprising 20 to 23 base pairs. The produced miRNA is incorporated into a protein complex called RNA-induced silencing complex (RISC), binds mainly to target mRNA having complementary sequence in the 3′ untranslated region and suppresses the expression of target mRNA through the activity of Argonaute acting as a functional domain of RISC. One miRNA has several hundreds kinds of mRNA complementary sequences in the 3′ untranslated region and miRNA is able to regulate a plurality of gene expressions at the same time. It has been becoming clear that some miRNA shows a tissue-specific expression mode, participates in generation of tissues and organs thereof and involves in various biofunction and development of disease such as immunity, oncogenesis, aging, etc.
miR-140 is one of miRNA which has been reported to express specifically to the cartilage forming region in a developing period of mice and is coded to intron 16 of the gene of WW domain containing E3 ubiquitin protein ligase 2 (Wwp2). miR-140 specifically rises its expression together with cartilage differentiation from mesenchymal cells. miR-140 also plays an important role in differentiation and homeostasis maintenance of cartilage in, for example, such a manner that a miR-140-deleted knockout mouse results in abnormality in chondrocyte growth in endochondral ossification. Moreover, in osteoarthritis which is a cartilage degenerative disease in a joint, expression of miR-140 in chondrocytes specifically decreases. In view of the fact that a miR-140 knockout mouse shows a morbid state like osteoarthritis and that a transgenic mouse where miR-140 is excessively expressed shows a resistance to osteoarthritis, miR-140 is deeply participated in occurrence of cartilage degenerative disease. Accordingly, a drug exhibiting the activity of regulating the expression of miR-140 is expected as a therapeutic agent for cartilage degenerative diseases such as osteoarthritis or intervertebral disk degeneration. However, due to the reason that the regulation mechanism of miR-140 is not clear, it has been difficult to screen a drug having the activity of regulating its expression.
Incidentally, Sox9 which is one of transcription factors plays an important role for controlling the chondrocyte differentiation. During the chondrogenesis, Sox9 is expressed in all of chondrocyte progenitors and chondrocytes but the expression dramatically decreases in hypertrophic chondrocytes. In an expression regulatory region for structural gene such as type 2 collagen (Col2a1) or aggrecan (Agcl) which is a main constitute of cartilage, consensus sequence (A/T)(A/T) CAA (T/A) G to which Sox9 binds is present and, when Sox9 binds to this region, expression of each gene is induced and cartilage differentiation is promoted. Moreover, although it has been known that, in cartilage differentiation, L-Sox5 and Sox6 which are other transcription factors work cooperatively with Sox9 to promote the cartilage differentiation, genes which are targets thereof have not been made clear except Col2a1 and Agcl and the regulatory mechanism where they potentiate the function of Sox9 has not been made clear yet.
In Non-Patent Document 1, Yang et al report that transcription start site for pri-miR140 exists in intron 10 of Wwp2 gene, that a luciferase reporter vector is constructed using a 2 kbp upstream region of pri-miR140 and that Sox9 regulates miR-140 expression in the region. Yang et al further found two Sox9-binding sites in the region (CAGGTTCCTTTGT in −824 bp upstream region of pri-iR140 and TAAAGGTGCTTTGT in −200 bp upstream region thereof) and made clear that, when mutation is introduced into the binding site at −200 bp, the reporter activity significantly decreases. However, Non-Patent Document 1 does not mention the cooperative action of Sox9 with Sox5 and Sox6 and neither mentions nor even suggests to apply those binding regions and reporter vector binding to those regions for a drug screening. In addition, the DNA sequence of the regions other than the above-mentioned two Sox9-binding sites is not disclosed in Non-Patent Document 1.